Antifolates such as methotrexate were originally developed as valuable chemotherapy agents to treat hematopoietic malignancies. More recently, newer antifolates were developed for treating solid tumors. Pemetrexed (Pmx, Alimta) was approved in 2004 for treating non-small cell lung cancer and mesothelioma. While Pmx shows antitumor activity, this agent lacks tumor selectivity and induces toxicity. The latter has been attributed to intracellular membrane transport of Pmx by Reduced Folate Carrier (RFC). RFC is a ubiquitously expressed folate facilitative transport protein that is present in both tumor and normal cells such as bone marrow. Folate Receptors (FRs) are an alternative mechanism for internalizing (anti)folates. FR, unlike other folate transport proteins is only expressed basolaterally and is exposed to circulating blood in tumors, in contrast to its apical localizationin normal tissues. In conjunction with this abnormal cellular architecture in tumors, FR is also overexpressed in a variety of malignancies including those of the ovaries, uterus and brain. Approximately 90% of epithelial ovarian cancers have been reported to exhibit elevated expression of FR with receptor densities correlating to tumor grade and stage. I hypothesize that the differential expression of FR in solid (ovarian) tumor cells enables tumor targeting of novel cytotoxic antifolates, reflecting selectivity for cellular uptake by FR over RFC. I further hypothesize that novel FR antifolate substrates that inhibit de novo purine nucleotide biosynthesis show tumor selectivity due to the absence of purine salvage and/or indirectly target downstream AMPK and mTOR signaling pathways. Antifolate therapeutics directed to FR and to specific folate-dependent intracellular targets will induce selective killing of tumor cells and reduce toxicity. To investigate this hypothesis, I propose the following specific aims: (Aim 1) to develop novel solid tumor-targeted antifolate therapeutics with selective membrane transport by FR over RFC that target de novo purine nucleotide biosynthesis and downstream signaling pathways (e.g., AMPK, mTOR); and (Aim 2) to identify determinants of cytotoxic activity and selectivity for the aforementioned solid tumor targeted agents in in vitro and in vivo model systems. To complete these aims, I will screen a series of rationally designed folate analogs and determine their antiproliferative and cytotoxic potencies via purine nucleotide depletion and their preferred mechanisms of membrane transport. I will establish their detailed mechanisms including cellular metabolism, intracellular targets, and impact on downstream signaling pathways. I will determine cellular determinants of drug activity including the impact of purine salvage and the presence or absence of other folate transport systems on drug activity. Finally I will establish in vivo efficacies within the broader context of these cellular determinants. Curren treatment strategies for late stage ovarian cancer are often ineffective and are associated with toxic side effects. Hence, there is a compelling rationale for developing newer FR-targeted therapies for treating ovarian carcinomas.